An Oxalate Transporter Gene, AtOT, Enhances Aluminum Tolerance in Arabidopsis thaliana by Regulating Oxalate Efflux

Secretion and efflux of oxalic acid from roots is an important aluminum detoxification mechanism for various plants; however, how this process is completed remains unclear. In this study, the candidate oxalate transporter gene AtOT, encoding 287 amino acids, was cloned and identified from Arabidopsis thaliana. AtOT was upregulated in response to aluminum stress at the transcriptional level, which was closely related to aluminum treatment concentration and time. The root growth of Arabidopsis was inhibited after knocking out AtOT, and this effect was amplified by aluminum stress. Yeast cells expressing AtOT enhanced oxalic acid resistance and aluminum tolerance, which was closely correlated with the secretion of oxalic acid by membrane vesicle transport. Collectively, these results underline an external exclusion mechanism of oxalate involving AtOT to enhance oxalic acid resistance and aluminum tolerance.


Introduction
In acidic soil (pH < 5), active AlOH 2+ and Al 3+ ions will be adsorbed on the cation exchange sites on the surface of soil particles or dissolved into the soil solution, thereby causing aluminum toxicity and harmful effects on plant growth and soil microbial activities [1][2][3]. With the deterioration of the soil environment and the application of chemical fertilizers, the degree of soil acidification is further deepened and the gross area of soil acidification is further expanded, thereby making aluminum toxicity a non-negligible factor in crop production [4]. Therefore, it is vital to study the mechanism of aluminum toxicity and detoxification in plants, which is one of the hotspots of plant stress biology [5,6].

Expression Patterns of AtOT
Quantitative real-time PCR (qRT-PCR) was used to determine the transcript profi of AtOT. For Al 3+ stress, the Arabidopsis seedlings with two true leaves were treated wi AlCl3 . 6H2O on 1/2 MS solid medium ( Figure 2A). As shown in Figure 2B, AtOT presente a constitutive expression pattern, which was richest in the root, followed by the stem, an lowest in the leaf. Following treatment with different concentrations of Al 3+ , expressio levels of AtOT showed a significant upregulation from 50 μM Al 3+ to 200 μM Al 3+ an peaked at 100 μM Al 3+ with an approximately 4.51-fold increase in expression ( Figure 2C In addition, AtOT expression gradually increased under the time-course treatments of 10 μM Al 3+ with prolonged treatment and reached its highest value at 48 h ( Figure 2D).

Subcellular IzationLocalization of AtOT
The 35S::AtOT-GFP (green fluorescent protein) recombinant was transformed into the tobacco leaf epidermal cells for the subcellular localization of the AtOT protein. Observation by an LSM800 confocal laser scanning microscope revealed that 35S::AtOT-GFP was restricted to the plasma membrane only ( Figure 3) thereby indicating that AtOT is localized in the plasma membrane in contrast to 35S::1300-GFP alone, which exhibited fluorescence throughout the cell.

Reverse Genetic Analysis of AtOT in Arabidopsis
The phenotype, root length, proline content, and MDA content of wild-type Col-0 and an atot mutant under different concentrations of Al 3+ stress were compared to examine whether or not Al-responsive AtOT is involved in aluminum detoxification. The homozygosity of the atot mutant was firstly confirmed. As shown in Figure 4A, the five atot mutant plants showed clear band for LBb1.3 + RP primers but no clear band for LP + RP primers in the PCR; therefore, they were all homozygotes. However, since plant no. 2 and no. 3 showed weak bands for LP + RP primers, to make the experiments more convincing, only the seeds produced from plants no. 1, 4, and 5 were collected as atot mutant homozygotes and used for the subsequent experiments. The root lengths of the wild-type and atot mutant were 4.03 and 2.74 cm, respectively, under non-stressed conditions, which reflected that knocking out AtOT might negatively affect root growth. With the increase in Al 3+ concentration, the root length of the wild type and atot mutant showed a trend of shortening and finally reached the lowest values at 200 µM Al 3+ , which were 1.32 and 0.70 cm, respectively ( Figure 4B,C), indicating that aluminum stress was more harmful to the atot mutant than to the wild-type. We also measured the contents of proline (Pro) and malondialdehyde (MDA) in the wild type and atot mutant under aluminum stress to verify this conclusion. The contents of Pro and MDA in the wild type and atot mutant gradually increased with the increase in Al 3+ concentration, and these contents in the atot mutant were significantly higher than those in the wild type under the same concentration of Al 3+ , which proved the positive role of AtOT in preventing aluminum-toxicity-induced oxidative damage ( Figure 4D,E).

Expression Patterns of AtOT
Quantitative real-time PCR (qRT-PCR) was used to determine the transcript of AtOT. For Al 3+ stress, the Arabidopsis seedlings with two true leaves were treate AlCl3 . 6H2O on 1/2 MS solid medium ( Figure 2A). As shown in Figure 2B, AtOT pre a constitutive expression pattern, which was richest in the root, followed by the ste lowest in the leaf. Following treatment with different concentrations of Al 3+ , exp levels of AtOT showed a significant upregulation from 50 μM Al 3+ to 200 μM A peaked at 100 μM Al 3+ with an approximately 4.51-fold increase in expression (Figu In addition, AtOT expression gradually increased under the time-course treatment μM Al 3+ with prolonged treatment and reached its highest value at 48 h (Figure 2D

Functional Characterization of AtOT in Yeast
AtOT was expressed in the yeast mutant AD1-8 to investigate the function of AtOT in oxalic acid transportation. Moreover, the construct pDR196-FpOAR and empty vector pDR196 were used as positive and negative controls, respectively ( Figure 5A). Based on the colony phenotype, the yeast strain AD1-8 can grow normally if the concentration of oxalic acid is not higher than 2 mM, which depends on the autologous oxalic acid resistance. Meanwhile, on plates containing 4-8 mM oxalic acid, the AtOT-transformed yeast showed clear cell growth which was similar to the positive control, whereas very little growth was observed in the negative control. When the concentration of oxalic acid reached 10 mM, the AtOT-transformed yeast could still grow, whereas it was hard for the negative and positive controls to form any colonies. On the other hand, the qRT-PCR showed that AtOT expression could be induced by oxalic acid stress and it peaked at 2 mM oxalic acid in yeast cells ( Figure 5B). These results implied that AtOT enhanced yeast cell oxalic acid resistance through some pathways responding to oxalic acid.
the bars indicate significant differences among the treatments at p < 0.05.

Subcellular izationLocalization of AtOT
The 35S::AtOT-GFP (green fluorescent protein) recombinant was transformed the tobacco leaf epidermal cells for the subcellular localization of the AtOT protein servation by an LSM800 confocal laser scanning microscope revealed that 35S::AtOT was restricted to the plasma membrane only ( Figure 3) thereby indicating that AtO localized in the plasma membrane in contrast to 35S::1300-GFP alone, which exhi fluorescence throughout the cell.

Reverse Genetic Analysis of AtOT in Arabidopsis
The phenotype, root length, proline content, and MDA content of wild-type C and an atot mutant under different concentrations of Al 3+ stress were compared to exa whether or not Al-responsive AtOT is involved in aluminum detoxification. The hom gosity of the atot mutant was firstly confirmed. As shown in Figure 4A, the five atot mu plants showed clear band for LBb1.3 + RP primers but no clear band for LP + RP pri in the PCR; therefore, they were all homozygotes. However, since plant no. 2 and showed weak bands for LP + RP primers, to make the experiments more convincing, the seeds produced from plants no. 1, 4, and 5 were collected as atot mutant homozyg and used for the subsequent experiments. The root lengths of the wild-type and atot tant were 4.03 and 2.74 cm, respectively, under non-stressed conditions, which refle that knocking out AtOT might negatively affect root growth. With the increase in Further yeast transformants were cultured in SD (-Ura) liquid medium containing 2 mM oxalic acid for 13 days. Although the pH value of each transformant decreased with time, the pH value and trend of the positive control and the AtOT-transformed yeast were the same and the pH value of the negative control was always higher than that of the positive control and the AtOT-transformed yeast under the same culture days (Table 1). At the end of the culture, the pH of each transformant reached the lowest value, with the average pH for the positive control, the AtOT-transformed yeast, and the negative control reaching 2.42, 2.38, and 2.76, respectively. Meanwhile, the dry weight of each transformant increased with time, and the overall dry weight was as follows: AtOT-transformed yeast > positive control > negative control ( Table 2). AtOT may affect the oxalic acid metabolism of yeast cells and reduce the inhibition of oxalic acid to yeast cells; hence, the acidity of the bacterial solution significantly decreased and the biomass accumulation increased under oxalic acid stress.
The oxalic acid content in yeast cells was determined by the sulfosalicylic acid method. Oxalic acid stress caused the oxalic acid content in yeast to significantly increase in a short time (≤1 day), thereby indicating that the short-term harmful effect of oxalic acid stress on yeast cells was inevitable. During the 1st to 13th day of culture, the oxalic acid content of the positive control and the AtOT-transformed yeast showed a decreasing trend over time ( Figure 6A). Finally, the oxalic acid content of the positive control and AtOT-transformed yeast was 4.12 mM and 5.83 mM, respectively. However, the oxalic acid content of the negative control was always at a high level. At the end of the culture, it was significantly higher than that of the positive control and AtOT-transformed yeast, with it being 11.20 mM.
concentration, the root length of the wild type and atot mutant showed a trend of shortening and finally reached the lowest values at 200 μM Al 3+ , which were 1.32 and 0.70 cm, respectively ( Figure 4B,C), indicating that aluminum stress was more harmful to the atot mutant than to the wild-type. We also measured the contents of proline (Pro) and malondialdehyde (MDA) in the wild type and atot mutant under aluminum stress to verify this conclusion. The contents of Pro and MDA in the wild type and atot mutant gradually increased with the increase in Al 3+ concentration, and these contents in the atot mutant were significantly higher than those in the wild type under the same concentration of Al 3+ , which proved the positive role of AtOT in preventing aluminum-toxicity-induced oxidative damage ( Figure 4D,E).

Functional Characterization of AtOT in Yeast
AtOT was expressed in the yeast mutant AD1-8 to investigate the function of AtOT in oxalic acid transportation. Moreover, the construct pDR196-FpOAR and empty vector pDR196 were used as positive and negative controls, respectively ( Figure 5A). Based on the colony phenotype, the yeast strain AD1-8 can grow normally if the concentration of oxalic acid is not higher than 2 mM, which depends on the autologous oxalic acid resistance. Meanwhile, on plates containing 4-8 mM oxalic acid, the AtOT-transformed yeast showed clear cell growth which was similar to the positive control, whereas very little growth was observed in the negative control. When the concentration of oxalic acid reached 10 mM, the AtOT-transformed yeast could still grow, whereas it was hard for the negative and positive controls to form any colonies. On the other hand, the qRT-PCR showed that AtOT expression could be induced by oxalic acid stress and it peaked at 2 mM oxalic acid in yeast cells ( Figure 5B). These results implied that AtOT enhanced yeast cell oxalic acid resistance through some pathways responding to oxalic acid. Further yeast transformants were cultured in SD (-Ura) liquid medium containing 2 mM oxalic acid for 13 days. Although the pH value of each transformant decreased with time, the pH value and trend of the positive control and the AtOT-transformed yeast were the same and the pH value of the negative control was always higher than that of the positive control and the AtOT-transformed yeast under the same culture days (Table 1).   In addition, the oxalic acid content in the culture medium was determined using the same method as described above. As shown in Figure 6B, the oxalic acid content in the medium of the negative control changed irregularly during the culture time, which showed that oxalic acid stress had caused disorder of oxalic acid metabolism in the negative control. Meanwhile, with the passage of culture time, the oxalic acid content in the medium of the positive control and AtOT-transformed yeast generally showed an upward trend and two common points between them that deserve to be noticed. The first common point was that the oxalic acid content in the medium rapidly increased at a certain time point, and the second common point was that the oxalic acid content in the medium was relatively stable for a period after the rapid increase, which further indicated that AtOT may be functionally related to oxalic acid efflux.
The [ 13 C]oxalic acid residue in the filtrate of yeast membrane vesicles was recorded in the form of δ 13 C by in an vitro oxalate transport study, which indirectly reflected the absorption of oxalic acid by vesicles. Apparent differences were observed between membrane vesicles of AtOT-transformed yeast and the negative control whether MgATP was added or not (Figure 7). The δ 13 C in the filtrate of the negative control was 5.72 times more than that in the filtrate of the AtOT-transformed yeast membrane vesicles when lacking MgATP. In the presence of MgATP, the magnification was increased to 6.01, thereby suggesting that AtOT significantly regulated the absorption of oxalic acid by membrane vesicles.
The involvement of AtOT in aluminum tolerance was identified by the same method as the oxalic acid stress study. The empty vector pDR196 was used as the negative control in this study as well. At an Al 3+ concentration below 2.7 mM, the growth of the negative control and the FpOARand AtOT-transformed yeast did not significantly differ. Nevertheless, it was hard for the negative control to form a colony once the Al 3+ concentration reached 2.7 mM; on the contrary, the AtOT-transformed yeast could still grow ( Figure 8A). When examining the transcripts of AtOT, the amount of AtOT transcripts increased 1.58-, 2.26-, 3.15-, and 3.18-fold as compared to that of the control under 2.4, 2.6, 2.7, and 2.8 mM Al 3+ stresses, respectively ( Figure 8B). These results revealed that AtOT improved the aluminum tolerance of yeast cells under high concentrations of aluminum.
The growth of each transformant was consistent with the characteristics of the Sshaped growth curve ( Figure 8C). The FpOAR-transformed yeast and the negative control showed a similar pattern in the growth trend, and their OD 600 values were finally stable at about 2.10 and 2.24, respectively. Nevertheless, the AtOT-transformed yeast entered the logarithmic growth phase at 8 h and reached the platform stage at 18-20 h, with an OD 600 value of approximately 1.83 at the end of the culture. Although the OD 600 value of AtOT-transformed yeast was slightly lower than that of the negative control and FpOARtransformed yeast at the platform stage, the time that it entered the logarithmic growth phase was much faster than that of the negative control and FpOAR-transformed yeast which reflected its strong adaptability to aluminum stress. These results implied that AtOT might confer the aluminum tolerance to the recombinant yeast cell. The [ 13 C]oxalic acid residue in the filtrate of yeast membrane vesicles was recorded in the form of δ 13 C by in an vitro oxalate transport study, which indirectly reflected the absorption of oxalic acid by vesicles. Apparent differences were observed between membrane vesicles of AtOT-transformed yeast and the negative control whether MgATP was  The involvement of AtOT in aluminum tolerance was identified by the same meth as the oxalic acid stress study. The empty vector pDR196 was used as the negative contr in this study as well. At an Al 3+ concentration below 2.7 mM, the growth of the negati control and the FpOAR-and AtOT-transformed yeast did not significantly differ. Nev theless, it was hard for the negative control to form a colony once the Al 3+ concentrati reached 2.7 mM; on the contrary, the AtOT-transformed yeast could still grow ( Figure 8A When examining the transcripts of AtOT, the amount of AtOT transcripts increased 1.5 , 2.26-, 3.15-, and 3.18-fold as compared to that of the control under 2.4, 2.6, 2.7, and 2 mM Al 3+ stresses, respectively ( Figure 8B). These results revealed that AtOT improved t aluminum tolerance of yeast cells under high concentrations of aluminum.
The growth of each transformant was consistent with the characteristics of the shaped growth curve ( Figure 8C). The FpOAR-transformed yeast and the negative contr showed a similar pattern in the growth trend, and their OD600 values were finally stab at about 2.10 and 2.24, respectively. Nevertheless, the AtOT-transformed yeast entered t logarithmic growth phase at 8 h and reached the platform stage at 18-20 h, with an OD value of approximately 1.83 at the end of the culture. Although the OD600 value of AtO transformed yeast was slightly lower than that of the negative control and FpOAR-tran formed yeast at the platform stage, the time that it entered the logarithmic growth pha was much faster than that of the negative control and FpOAR-transformed yeast whi reflected its strong adaptability to aluminum stress. These results implied that AtO Some soluble proteins will chelate with metal ions under metal ion stress to reduce the toxic effects caused by metal ions. Therefore, the total protein (TP) content was measured as an indicator of the degree of toxicity of aluminum stress to cells ( Figure 8D). The TP content of each transformant was similar under normal conditions. However, the TP content of the negative control significantly decreased and was lower than that of the FpOARand AtOT-transformed yeasts under 2.7 mM Al 3+ stress, with it being 9014.5 µg/mL, and the TP content of the FpOARand AtOT-transformed yeasts decreased slightly, which were 10,327.6 µg/mL and 10,477.2 µg/mL, respectively. These results indicated that the adverse effect of Al 3+ to the FpOARand AtOTtransformed yeasts was lesser than that of the negative control.
Malondialdehyde (MDA) is a product of membrane lipid peroxidation, and its content can be used to measure the resistance of yeast cells to various stresses. In general, the higher the intracellular MDA content, the higher the degree of plasma membrane damage. The MDA content of each transformant was similar when there was no aluminum stress. Under 2.7 mM Al 3+ stress, the MDA content of the negative control was significantly higher than that of the FpOARand AtOT-transformed yeasts, thereby reaching 0.172 µM, which was 2.11 times higher than that under normal conditions. In contrast, the MDA content of the FpOARand AtOT-transformed yeasts increased slightly, thereby reaching 0.089 µM and 0.104 µM, respectively, which were 1.12 times and 1.26 times higher than that under normal conditions ( Figure 8E). These results indicated that the oxidative damage of the plasma membrane in FpOARand AtOT-transformed yeasts was less than that in the negative control under aluminum stress.
Peroxidase (POD) is widely found in animals, plants, and microorganisms. It is a marker enzyme of peroxisomes that plays an important role in physiological processes such as scavenging reactive oxygen species (ROS), disease resistance, salt resistance, and cell death. No significant difference was observed in POD content among different trans-formants under normal conditions, as shown in Figure 8F. Under 2.7 mM Al 3+ stress, the POD content of the negative control increased to 0.961 U/mg protein, which was significantly higher than that of the FpOARand AtOT-transformed yeasts and was 2.05 times of that under normal conditions. However, the POD content of FpOAR-transformed yeast increased to 0.71 U/mg protein, which was 1.43 times of that under normal conditions. The POD content of the AtOT-transformed yeast did not change significantly compared with that under normal conditions, which was 0.445 U/mg protein. These results suggest that the ROS content and oxidative damage degree in the negative control were higher than those of the FpOARand AtOT-transformed yeasts, which further proved that AtOT can detoxify aluminum.

Discussion
The mechanism of roots secreting organic acids to detoxify aluminum has been studied in various plants. Among them, malic AtMATE and citric acid transporter genes AtALMT1 of the model plant Arabidopsis guide help in the identification of organic acid transporter genes in other plants. However, the oxalate transporter gene in Arabidopsis has not been identified and reported until now. The candidate oxalate transporter gene AtOT

Discussion
The mechanism of roots secreting organic acids to detoxify aluminum has been studied in various plants. Among them, malic AtMATE and citric acid transporter genes AtALMT1 of the model plant Arabidopsis guide help in the identification of organic acid transporter genes in other plants. However, the oxalate transporter gene in Arabidopsis has not been identified and reported until now. The candidate oxalate transporter gene AtOT was obtained from Arabidopsis through BLASTP homologous alignment. We performed bioinformatics analysis and investigated the effects of AtOT on oxalic acid secretion and aluminum tolerance in yeast and Arabidopsis to verify the function of AtOT.
The prediction analysis of the conserved domain showed that the AtOT protein had SNARE-assoc conserved domains, belonging to the SNARE superfamily. Unlike MATE and ALMT proteins which are only involved in a few specific substrates such as citric acid and malic acid, the transport types and substrates of SNARE proteins are very complex [43,44]. At present, the research on plant SNARE proteins is still in its infancy, many scholars believe that the SNARE proteins primarily play a role in the transport of the inner membrane system by membrane fusion, including regulating vesicle synthesis, directional transport, and identifying and promoting the fusion between vesicles and specific target membranes which are highly conserved in animals, plants, and fungi [45]. According to our previous research, AtOT is quite different from other identified plasma-membrane-localized plant SNARE proteins. It was clustered into the SNARE-assoc subfamily with Hevea brasiliensis oxalate transporter HbOT1 and HbOT2 [42]. Combined with the results of subcellular localization and function characterization in yeast, we concluded that the AtOT protein may play a role in the membrane vesicle transport of certain substances such as oxalic acid. Moreover, the AtOT protein, located in the plasma membrane, has five transmembrane helices and has a larger hydrophobic part than the hydrophilic part, which is consistent with the basic characteristics of transporters and functional annotation of SNARE proteins.
The expression of AtOT in Arabidopsis wild-type Col-0 was significantly upregulated under aluminum stress, which increased first and then decreased with the increase in aluminum concentration and increased with the aluminum treatment time, suggesting that AtOT is inducible by active Al 3+ ions. Meanwhile, the knockout of AtOT hurt the growth of the roots, and aluminum stress amplified the adverse effect. Therefore, AtOT may play an important role in root growth and is closely related to the mechanism of aluminum detoxification in Arabidopsis.
The oxalic acid resistance and aluminum tolerance of AtOT was identified by the yeast AD1-8 system. On the colony phenotype, the AtOT-transformed yeast showed stronger oxalic acid resistance and aluminum tolerance than the negative control under oxalic acid and aluminum stress. The lower pH value of the culture medium and higher dry weight of the yeast cells indicated that the AtOT-transformed yeast had better adaptability than the negative control in the liquid environment with 2 mM oxalic acid stress. The similar trend of the pH value and faster growth rate to the positive control FpOAR revealed the possibility of AtOT as an oxalate transporter. Based on the determination of oxalic acid content in vitro and vivo, we found that the AtOT and positive control FpOAR could help yeast cells overcome the continuous injury caused by oxalic acid stress and improve the oxalic acid metabolism rule of yeast cells under oxalic acid stress. The similar change in oxalic acid contents indicates that AtOT may play an important role in the transport and efflux of oxalic acid. The in vitro incubation of yeast membrane vesicles with [ 13 C]oxalic acid verifying that AtOT regulates the absorption of oxalic acid by membrane vesicles, which further confirmed that AtOT is a SNARE family protein that could regulate the transport and efflux of oxalate by the membrane vesicle transport in Arabidopsis. Meanwhile, AtOTtransformed yeast entered the platform stage earlier under aluminum stress as compared with the negative control and FpOAR-transformed yeast, thereby reflecting its stronger adaptability to aluminum stress. All these results suggest that AtOT may act as an oxalic acid transporter involved in the Arabidopsis aluminum detoxification.
Total protein and POD contents are important indices to measure the degree of oxidative damage in cells, while MDA content is an important index to measure the degree of oxidative damage in the plasma membrane. Correspondingly, they are used to examine the oxidative damage of yeast cells by Al 3+ in this study. Al 3+ inevitably caused the oxidation and destruction of the plasma membrane by binding to membrane proteins, competing for receptor binding sites, and seizing ion channels. If AtOT primarily detoxifies aluminum from the inside of the cell by the compartmentation of vacuoles or the production of specific proteases [46], it will lead to an increase in MDA content. The total protein content, POD, and MDA in the AtOT-transformed yeast were more stable than those in the negative control before and after aluminum stress. Hence, we concluded that AtOT can significantly reduce aluminum toxicity in vivo and plasma membranes and may play an important role in the external exclusion mechanism of aluminum detoxification by regulating the efflux of oxalic acid.

Materials and Treatments
The homozygous Arabidopsis atot mutant (SALK_002559C), in which the AtOT gene is knocked out, was purchased from the AraShare Arabidopsis mutant library (http://www. arashare.cn/index/ (accessed on 10 November 2021)), and the Arabidopsis wild type Col-0 was used in this study as the control.
The seeds were sterilized in 75% ethanol for 2 min, 95% ethanol for 2 min, and 10% NaClO for 10 min and washed five times with ddH 2 O before culture. Then, the disinfected seeds were sown on 1/2 MS solid medium, vernalized at 4 • C for 48 h in the dark, and then cultured in a light incubator. The light incubator program was set to 22 • C, a relative humidity of 70%, and a photoperiod of 16 h/8 h (day/night). After growing two true leaves, the seedlings were transferred to 1/2 MS solid medium (pH = 4.2) containing Al 3+ . The wild-type Col-0 seedlings treated with 0 (CK), 25, 50, 100, 150, and 200 µM Al 3+ for 48 h and treated with 100 µM Al 3+ for 0 (CK), 3, 6, 12, 24, and 48 h were collected to extract RNA for expression analysis of AtOT in response to the concentration and stress time of aluminum, respectively.
The yeast mutant strain AD12345678 (AD1-8) was donated by Professor Tang of Shanghai JiaoTong University and Professor Richard Cannon of Otago University.

RNA Extraction, cDNA Synthesis, and qRT-PCR Analysis
Total RNA was extracted from the seedlings of Arabidopsis using a TIANGEN plant total RNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China) and cDNA was synthesized using TaKaRa reverse transcription kit (Baori Doctor Technology Co., Ltd., Dalian, China). The concentration and purity of RNA and cDNA were analyzed by a NanoDrop 2000 ultra-micro nucleic acid protein analyzer (Thermo Fisher Technology (China) Co., Ltd., Shanghai, China). The qRT-PCR analysis was performed on a CFX96 TOUCH real-time fluorescent quantitative PCR instrument (Bio-Rad, Hercules, CA, USA). The internal reference gene AtActin2 (AT3G18780) was selected to normalize the data. The quantitative variations of gene expression were analyzed by the 2 −∆∆CT method and the specific primers are listed in Supplementary Table S1.

Cloning and Bioinformatics Analysis
The ORF sequences of AtOT were amplified by PCR using specific primers (Supplementary Table S1) with the cDNA of Arabidopsis wild-type Col-0 as a template and the fragment was connected to the pMD-18T vector for sequencing.

Subcellular Localization of AtOT in N. benthamiana
In the subcellular localization assay, the cDNA of AtOT was subcloned into the expression vector pCAMBIA1300:GFP under the control of the 35S promoter using the specific primers listed in Supplementary Table S1. The pCAMBIA1300:AtOT-GFP fusion vector and pCAMBIA1300:GFP were separately introduced into the Agrobacterium tumefaciens strain GV3101 by the heat shock method. The A. tumefaciens suspensions were subsequently injected into leaves of 4-week-old Nicotiana benthamiana plants and the transient expression of AtOT was detected after 48 h using LSM800 a confocal laser scanning microscope (Carl Zeiss Shanghai Co. Ltd., Shanghai, China).

Homozygous Identification and Reverse Genetic Analysis of AtOT in Arabidopsis
To confirm the atot mutant homozygotes, total DNA was extracted by the CTAB method from the different atot mutant plants cultured for 3 weeks. Then, the three-primer method, i.e., the LP, RP, and LBb1.3 primers (Supplementary Table S1) designed by the Genomic Analysis Laboratory of the Salk Institute (http://signal.salk.edu/ (accessed on 7 January 2022)), was utilized for the identification of the atot mutant homozygous plants. The plants which have a clear band for the LBb1.3 + RP but have no band for the LP + RP primers during PCR were identified to be homozygous atot mutants and utilized in the subsequent experiments [47]. The Col-0 and atot mutant seedlings under 0 (CK), 25, 50, 100, 150, and 200 µM Al 3+ stresses for 2 weeks were randomly selected for root length recording and the determination of proline (Pro) and malondialdehyde (MDA). Pro content was determined using the proline quantitative assay kit (Nanjing Jiancheng Bioengineering Institute Co., Ltd., Nanjing, China). MDA content was determined by TBA colorimetry [48].
Transformants cultured with OD 600 = 1.0 were extracted and added in SD (-Ura) liquid medium containing 2 mM oxalic acid and cultured at 30 • C with 180 rpm for 13 days to further study the oxalic acid resistance of AtOT. Subsequently, the bacterial solution was centrifuged at 1000× g for 10 min to separate the yeast cells from the medium after being washed twice with ddH 2 O to determine the oxalic acid content in the yeast cells and the medium using boxbio oxalic acid (OA) content determination kit (Box Biotechnology Co., Ltd., Beijing, China). The pH of the bacterial solution was determined by a METTLER TOLEDO FE plus pH meter (Metler Toledo Technology Co., Ltd., Shanghai, China). A SCIENTZ-30YG/A freeze-drying machine (Xinzhi Freeze-drying Equipment Co., Ltd., Ningbo, China) was used for freeze-drying the yeast cells.
Similarly, each transformant was cultured in a 50 mL SD (-Ura) liquid medium containing 2.7 mM Al 3+ for 48 h to further study the aluminum resistance mechanism of AtOT. The OD 600 value of the bacterial solution was measured every 2 h to plot the growth curve. Then, the bacterial solution was centrifuged at 1000× g for 10 min, and yeast cells were collected to determine the total protein (TP), malondialdehyde (MDA), and peroxidase (POD). TP and POD content were determined using a protein quantitative assay kit (Nanjing Jiancheng Bioengineering Institute Co., Ltd., Nanjing, China) and a peroxidase assay kit (Keming Biotechnology Co., Ltd., Suzhou, China), respectively.
Furthermore, vesicles (100 µg of protein) were incubated in 125 µL transport buffer (0.4 M glycerol, 100 mM KCl, 20 mM Tris-MES [pH = 7.4], 1 mM DTT, 0 (CK), or 5 mM MgATP) containing 0.2 mM [ 13 C]oxalic acid at 25 • C for 10 min, and the incubation solution was injected into syringe filters (33 mm in diameter and 0.45-µm pore size; Labgic Technology Co., Ltd., Beijing, China) and washed thrice with 1 mL transport buffer by needle-free injectors to absorb yeast vesicles. For the measurement of δ 13 C, 0.001 g silicon algae were added to 10 µL filtrate and then freeze-dried until the water was completely removed and transferred to a tin cup (6 mm × 4 mm) by a Flash EA 1112 Automatic Element Analyzer (Thermo Fisher Scientific Co., Ltd., Shanghai, China).

Statistical Methods
The gene expression level was from three biologicals with three technical repetitions. In addition, the pH value, dry weight, oxalic acid content, total protein content, malondialdehyde content, and peroxidase content of the yeast were the mean ± standard deviation of three biological replicates. Moreover, Microsoft Office Excel was used for data analysis and mapping. IBM SPSS Statistics 25 was used for a single-factor ANOVA test to analyze the difference. Duncan's method was used for the analysis.

Conclusions
In this study, the candidate oxalate transporter gene AtOT was cloned and identified from Arabidopsis. This gene can make yeast cells obtain stronger oxalate resistance, and we believe that this phenomenon is caused by its induction of oxalic acid efflux. On the other hand, it can promote the yeast aluminum tolerance to higher concentrations and can be induced by aluminum stress in Arabidopsis. It can regulate the membrane vesicle transport of oxalate to avoid the oxidative damage of Al 3+ . The knockout of AtOT will inhibit the root growth of Arabidopsis under normal conditions, and this inhibition will be further amplified under aluminum stress. All of these results suggest that AtOT likely played a pivotal role in regulating oxalate-related vesicle transport and the mechanism of oxalate efflux to detoxify aluminum; more solid research is worthwhile for the AtOT gene.